Abstract
Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.
Highlights
PURIFICATION AND COMPARISON OF LIGAND BINDING CHARACTERISTICS WITHTHAT OF ESCHERICHIA COLI-DERIVED RAT LIVER FATTY ACID-BINDING PROTEIN*
Rat intestinal fattyacid-binding protein (I-FABP) is lism and/or compartmentalizationof fatty acids within an abundant, 15,124-Da polypeptide found in the cy- enterocytes
Tosol of small intestinal epithelicaellls.It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes
Summary
Strategy for Expression of Intestinal FABP cDNA-In a previous publication we have described the efficient expression of rat liver FABP in E. coli [18], usingthe pLvectors of Remautet al. [19]. A unique NcoI restriction site was introduced into pPLc245, in the region of the plasmid that contained the translation initiationcodon of the MS2 replicase gene (see Fig. 1and Ref. 19) This unique NcoI site provides twomethodsforlinking "coding sequences" tothe MS2 translational control signals [20].The 3' recessed termini left after digestion with NcoI may be "filled-in'' with the Klenow fragment of DNA polymerase I. InRtaetstinal and Liver FABPs Expressed in E. coli proteins synthesized during this period This value was not significantly different from the amount of radiolabeled IFABP which accumulates in cells containing aplasmid identical to pIFABPexp but lacking the transcription termination signals (1.8%).Pulse-chase experiments demonstrated that I-FABP is stable within the bacterial cells for at least 60 min (data not shown).
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